Project Summary Staphylococcus aureus is a major human pathogen that is the leading cause of infections worldwide. It is the causative agent of multiple life threatening disease conditions including bacteremia, infectious endocarditis, osteomyelitis, septic arthritis and prosthetic joint infection. A major complication to treatment is the fact that the bacterium can form biofilms and infect locations that are hard to reach with systemic antibiotic administration. In addition, diagnosis can often be difficult depending on the location of the infection, level of bacterial burden and potential involvement of other bacterial species in the infection. Furthermore, S. aureus infections are often more severe and management more complicated with greater number of interventions required compared to other bacterial infections. Therefore novel strategies for rapid and effective diagnosis and treatment of S. aureus infections have the potential to have significant clinical impact. This proposal outlines our plans to use small molecule active site probes to identify serine hydrolases that are expressed by S. aureus bacteria. We believe that secreted or surface exposed serine hydrolases are ideal targets for both novel small molecule anti- virulence agents and for imaging contrast agents that can be used to non-invasively visualize sites of infection in vivo. Our preliminary efforts have already identified 12 new uncharacterized serine hydrolases and we have confirmed that at least one of these is involved in some aspect of virulence in the host. Therefore our primary aims are to 1) Functionally characterize serine hydrolases in S. aureus to identify optimal targets for imaging and therapeutic agents 2) Develop small molecule contrast agents and inhibitors that selectively target serine hydrolases involved in pathogenesis 3) Validate hydrolase-specific imaging probes and inhibitors in a mouse model of infection. Ultimately, success in these aims will not only lead to identification and functional characterization of novel regulators of S. aureus pathogenesis but also to the validation of imaging tools and inhibitors that could then be advanced to clinical applications in the future. Ultimately, clinical validated imaging and therapy agents have the potential to impact detection, diagnosis and therapy monitoring as well as to provide new ways to treat infection.